Preparation of nucleic acids from a sample containing the nucleic acids is an important technique in the fields of biotechnology, clinical diagnosis and the like. For example, in the genetic recombination techniques, it is required to isolate both a vector DNA and DNA to be cloned, and in order to conduct a genetic screening for a hereditary disease or an oncogene, it is required to extract nucleic acids from leucocyte cells and the like in blood.
Nucleic acids do not generally exist as isolated molecules, but they exist, for example, in bacteria, cells, virus particles and the like, and enclosed in cell membranes or cell walls consisting of proteins, lipids and saccharides. Further, nucleic acids themselves form complexes with histone proteins and the like. In order to extract nucleic acids existing in such a state, operations are required for disrupting cell membranes or cell walls enclosing the nucleic acids, solubilizing proteins of the complexes by denaturation or decomposition to release the nucleic acids and extracting the released nucleic acids.
As a method for preparing nucleic acids from cells, a method is commonly applied which comprising treating a sample containing cells with SDS or proteinase K for solubilization, then denaturing and removing the proteins with phenol to purify the nucleic acids (Molecular Cloning, 2nd Edition, 9.16-9.23, Cold Spring Harbor Laboratory Press, 1989). However, this method requires time and labor, and therefore, a more convenient method has been desired.
As a more convenient method for isolating nucleic acids, for example, WO99/22021 discloses a method of using silica. In this method, cells are first treated with a chaotropic reagent to lyse the cells and thereby release nucleic acids. Then, the nucleic acids are adsorbed on a nucleic acid binding carrier comprising silica or a derivative thereof, and this carrier is washed with a chaotropic reagent or an organic solvent by centrifugation. Finally, the nucleic acids are eluted with a low salt buffer. Although this method is more convenient compared with the phenol method, the method has drawbacks of still comprising many steps and requiring a centrifugal operation. Further, the method uses a chaotropic salt or ethanol that strongly inhibits enzymatic reactions such as those of PCR, which raises drawbacks of necessity of completely removing these substances thereby operations become complicated and time consuming and the like.
A method of preparing nucleic acids from peripheral blood leucocytes using a cell adsorbing fiber aggregates such as leucocyte separation filters is disclosed in Japanese Patent Publication (KOKOKU) No. 8-24583 and Japanese Patent Unexamined Publication (KOKAI) No. 8-280384. In the method disclosed in Japanese Patent Publication No. 8-24583, blood is first passed through a leucocyte separation filter to absorb leucocytes in blood on the filter, and thereby the leucocytes are separated from the other components of blood. The filter is washed by passing TE (10 mM Tris, 1 mM EDTA, pH 7.6) thorough the filter to remove proteins such as hemoglobin. The leucocytes separated and washed as described above are frozen at, for example, −80° C., together with the filter and then thawed by being left at room temperature. Then, TE-mix (TE, 10 mM NaCl, 1.5 mM MgCl2, pH 7.5) was added to the filter to collect the leucocytes adsorbed on fibrous materials of the filter from the fibrous materials. However, Japanese Patent Publication No. 8-24583 describes that the operation of extracting genomic DNA from the collected leucocytes is conducted by the conventional method (phenol treatment). Specifically, the publication describes a method of adding a surfactant such as 10% sodium dodecylsulfate (SDS) and a protease (proteinase K) to the leucocytes, incubating the mixture at 65° C. for 15 hours, then adding an RNase (RNase A), incubating the mixture at 37° C. for 1 hour, then treating the resultant with a phenol reagent, and precipitating DNAs with ethanol.
Japanese Patent Unexamined Publication No. 8-280384 discloses a method of adsorbing nucleated cells and extracting nucleic acids or proteins in the nucleated cells. As the methods of extracting nucleic acids or proteins, a method of applying a cell lysis solution to superfine fiber aggregates on which the cells are bound to lyse the cells and a method of disrupting the cells are mentioned. The advantage of this method is that the adsorbed cells per se are can be disrupted or subjected to a lysis treatment without desorption of the adsorbed cells. Since the adsorbed cells are disrupted or lysed without desorption of the adsorbed cells, this method can be more conveniently performed compared with a method of desorbing adsorbed cells. However, the method of this publication also uses conventional methods without modification for purification of nucleic acids after the cell lysis, and the publication fails to disclose a novel method. Specifically, the superfine fiber aggregates adsorbing cells are first treated with purified water or a surfactant, and nucleic acids are purified from a lysis solution which passed through the superfine fiber aggregates by the usual phenol/chloroform method.
As described above, although use of a cell adsorbing fiber aggregates such as leucocyte separation filters in the methods of preparing nucleic acids from peripheral blood leucocytes has been conventionally known, those methods are those merely performing the steps of from separating leucocytes to extracting nucleic acids by using a filter, and they use available nucleic acid purification methods for the subsequent nucleic acid purification step. Therefore, the methods have drawbacks that the nucleic acid purification step becomes complicated, and thus requires a lot of time and labor.
As a more convenient method, WO00/21973 discloses a method of directly purifying nucleic acids from cells by using a filter. This method comprises the following steps: (1) a sample containing cells is first passed through a filter to allow the cells to adsorb on the filter, (2) the cells adsorbed on the filter are then lysed, (3) the lysate is filtered through a filter, (4) nucleic acids adsorbed on the filter are washed, and (5) the nucleic acids are finally eluted from the filter. The adsorbed nucleic acids are eluted by warming to a temperature of from 40 to 125° C., and the elution buffer has a pH in the range of 5 to 11, and may have a high or low salt concentration. The value A260/A280 of the purified nucleic acids is 1.8, and they can be used as a template for PCR. WO00/21973 refers to Whatman GF/D variant filters as examples of filters usable for the purpose of purification of nucleic acids, and Whatman GF/C filters as unusable examples. It is essential that filters suitable for this method do not trap the purified DNAs when the purified DNAs are passed through the filters. Further, in this method, when the cells are lysed and passed through the filter, the yield of DNAs is decreased by 80%, and therefore the method is not practically applicable. Moreover, when nucleic acids are prepared from blood by using this method, experimenters need to hemolyze erythrocytes before lysing leucocytes. In addition, such a method of adsorbing cells on a filter and then performing the purification as described above has a drawback that the filter should be chosen depending on a type of cells.
As a further convenient method overcoming these drawbacks, WO03/006650 discloses a method of using a nonwoven fabric to separate, amplify and detect nucleic acids. This method comprises the steps of first contacting a cell extract with a nonwoven fabric to adsorb nucleic acids in the cell extract on the nonwoven fabric, and then adding a solution for amplifying the nucleic acids to the nonwoven fabric to amplify the nucleic acids using the nucleic acids adsorbed on the nonwoven fabric as a template. By this method, nucleic acids can be purified on the nonwoven fabric with an extremely convenient method of contacting nucleic acids in a sample with the nonwoven fabric without using any special reagents. Further, the nucleic acids purified on the nonwoven fabric, per se, can be subjected to an amplification reaction by contacting the nucleic acids with a reaction mixture for nucleic acid synthesis, and detection of nucleic acids can be performed conveniently. However, this method has a drawback that when nucleic acids existing at comparatively low concentrations (1 to 100 pg/mL) is to be trapped, the trapping rate is significantly reduced by influence of proteins such as albumin and globulin coexisting in the sample.
Further, as an example of use of a divalent metal ion for isolating nucleic acids, for example, a method of using silica (WO99/22021) is known. In this method, cells are first lysed by a treatment with a chaotropic reagent to release nucleic acids. Then, the nucleic acids are adsorbed on a nucleic acid binding carrier comprising silica or a derivative thereof, and this carrier is washed with a chaotropic reagent or an organic solvent by centrifugation. Finally, the nucleic acids are eluted with a low salt buffer. In this method, in order to maintain immobilization of nucleic acids on the carrier at the time of the adsorption of the nucleic acids to the carrier, various kinds of salts containing magnesium are used as additives to be added to the solution.
However, this method does not use magnesium ions as a means for trapping nucleic acids in a liquid phase by immobilizing the ions on the substrate side, and does not use magnesium ions as a primary means for isolation of nucleic acids. A method of separating double-stranded nucleic acids from proteins by using a magnesium salt is disclosed in Japanese Patent Application No. 5-164841. This method uses a polymer containing an aromatic moiety substituted with hydroxyl group and having a pK less than 9 as a substrate, and a liquid phase containing nucleic acids and proteins is contacted with the polymer at pH 7 to 10 in the presence of monovalent or multivalent cations to bind proteins and single-stranded nucleic acids to the substrate and separate double-stranded nucleic acids not bound to the substrate from the liquid phase. This method does not comprise any step of denaturing the sample under a strongly alkaline condition at a pH of 12 or higher, and magnesium is not positively immobilized on the substrate as a nucleic acid trapping agent. Furthermore, in this method, magnesium ions are not bound to the substrate in the presence of nucleic acids under a strongly alkaline condition at a pH of 12 or higher.